Overexpression of endogenous aldehyde:ferredoxin oxidoreductase genes enhances ethanol biosynthesis in Clostridium...

Bioethanol is a promising renewable fuel that mitigates dependence on fossil resources. As a potential candidate for bioethanol production, the homoacetogen Clostridium autoethanogenum is capable of metabolizing diverse carbon substrates, such as syngas components (CO and/or CO₂/H₂) and xylose. Since acetate formation is energetically favored over ethanol synthesis in acetogens, reassimilation of acetate through the aldehyde:ferredoxin oxidoreductase (AOR) pathway offers a feasible approach to enhance ethanol production. To improve AOR activity, two endogenous aor genes (CAETHG_0092 and CAETHG_0102) were individually overexpressed in C. autoethanogenum under the control of two separate constitutive promoters, P_thl and P_pta, generating four engineered strains: Ca(pta92), Ca(pta102), Ca(thl92), and Ca(thl102). Fermentation performance of the engineered strains was evaluated with xylose as the substrate. Compared to the wild-type strain, all engineered strains showed less acetate accumulation and higher ethanol yield. Moreover, the highest ethanol yield, specific productivity, and ethanol-to-acetate molar ratio were achieved in strain Ca(pta102), reaching 3.19-fold, 2.86-fold, and 11-fold of those in the wild-type strain, respectively. Engineered strains also exhibited elevated AOR activity in crude extracts, ranging from 2.52 to 4.74 times that of the wild-type strain. Results demonstrated that overexpressing either of the endogenous aor genes shifted product distribution toward ethanol in C. autoethanogenum, which provides a promising metabolic engineering strategy to boost ethanol biosynthesis.